Bioaerosols, (e.g., airborne microorganisms) have both natural and anthropogenic sources. They are found in the workplace and in homes. High concentrations may occur in or around buildings with defective air handling or air-conditioning systems, in houses with domestic animals, in manufacturing operations in which metalworking fluids are used, in dairy or other operations in which animals are confined, in sites of sludge application, in recycling or composting plants, and in sewage plants. Unlike most common atmospheric aerosols, airborne microorganisms can cause diseases, and, along with other biological (e.g., dust mite allergens) and non-biological (e.g., diesel exhaust particles) aerosols can cause allergies and respiratory problems. Bioaerosols are also feared as potential biowarfare and terrorists agents.
Improved methods for measuring aerosols, particularly bioaerosols, are needed. Presently, methods that measure aerosol size distributions in real time provide almost no information about particle types and are not able to identify specific microorganisms. For most allergens, toxins, or microorganisms, culturing the sample or the use of specific protein or nucleic acid recognition molecules is required.
There are presently several methods under development which, while not able to specifically identify bioaerosol particles, can run continuously and give an indication of the presence of biological aerosols. Several fluorescence particle counter devices that measure the elastic scattering and undispersed fluorescence of single aerosol particles as they are drawn through an optical cell have been developed. These devices have demonstrated promise for differentiating between biological and at least some nonbiological aerosols. However, they hold limited promise for classifying biological aerosols. Rather primitive techniques for measuring the fluorescence spectra of single aerosol particles have also been demonstrated. However, these techniques are not capable of measuring single particle spectra with a sufficient signal-to-noise ratio to be useful for classifying micrometer-sized biological particles.
In monitoring harmful bioaerosols a rapid response may be necessary in situations where it would be impractical to continuously run a sampler/identifier (a real time monitor might also suggest when to sample for specific harmful bioaerosols). Furthermore, recognition molecules are not always available for all particle types of interest (new bioaerosols may appear). In addition, some studies of bioaerosol dynamics and reactions (evaporation, growth, agglomeration, mixing, etc.) require real-time monitoring capability. Finally, in searching for or studying intermittent sources of bioaerosols, a rapid response may be advantageous. Despite the significant advancement in capabilities of the techniques referred to above, none of these methods are capable of measuring the fluorescence spectra of single micrometer-sized biological particles with a sufficient signal-to-noise ratio to classify them.
Building a point detector that exploits the intrinsic fluorescence of bioaerosol particles for their detection and classification is technically challenging for several reasons. Particles of interest may exist as a small concentration in a dominant background. Average fluorescence spectra accumulated for a population of aerosol particles may yield little or no information about the few particles of interest (i.e., single-particle spectra are required).
In addition, fluorescence signals are weak because single particles contain only a few picograms of material, and only a small fraction of the mass of biological particles is comprised of fluorophors.
Particles are generally dispersed nonuniformly in the air (their concentration fluctuations follow the Kolmogorov spectrum of atmospheric turbulence), and they must be detected at random times as they are carried rapidly by a stream of air through an optical cell.
Moreover, an optimal detector should excite particles in the ultraviolet where most biological particles (and biological molecules) fluoresce efficiently. Ultraviolet laser sources are costly and have relatively low energy output.
Bioaerosols of interest, including individual particles in bioaerosols, may be complex mixtures. Fluorescence from various components of the mixture may limit the usefulness of classification schemes. If fluorescence emission bands were narrow and the number of possible materials in a single particle were small, then it would likely be possible to solve the inverse problem and determine the materials that contributed to the spectrum.
However, the intrinsic fluorescence bands from biological materials tend to be spectrally wide; the primary fluorophors in the majority of bioaerosols fall into only a few broad categories (e.g., the aromatic amino acids, tryptophan, tyrosine, and phenylalanine; nicotinamide adenine dinucleotide compounds (NADH); flavins; and chlorophylls); and the number of possible materials is very large. The differences between spectra of bacteria appear to depend on preparation methods (growth media, type and extent of washing of the samples, etc.) more than they depend on intrinsic variations between well-purified bacteria.
Therefore, it may not be possible, except with severely restricted classes of bioaerosols, to identify specific bioaerosols based solely on their fluorescence spectra and their size (as determined from elastic scattering). The extent to which it may be possible to characterize naturally occurring and anthropogenically produced bioaerosols (e.g., group them into a few or even a few tens of categories) is yet unknown, however, it is expected that devices according to the invention described herein will provide highly reliable and rapid bioaerosol fluorescence spectra and particle size.
Optical techniques are used extensively for aerosol measurement. They are non-intrusive, provide essentially real-time data, and are relatively easy to use. Techniques for measuring aerosol scattering using nephelometers, aerosol absorption using photo-acoustics, aerosol extinction using tranmissometry, and aerosol size and concentration using light scattering particle counters, have matured significantly over the last 25 years.
In addition to advances in hardware (for example, advances in light sources, detectors, and computers), instrument response models for some of these techniques have been developed that put the interpretation of measured data on a sound footing, thereby making the techniques more definitive for aerosol measurement.
Of the techniques mentioned above, particle counters are one of the most widely used. They have been employed for determining estimates of the tropospheric and stratospheric aerosol burden, for monitoring concentrations of particles in clean rooms, and for detecting atmospheric aerosol pollutants. However, these instruments suffer from a critical limitation they provide almost no information about particle composition.
Detecting chemical composition of particles is desirable for a variety of applications, such as in detecting fugitive aerosol pollutants, differentiating between biological and non-biological aerosols (and classifying biological particles), or investigating aerosol drug-delivery systems.
Light scattering particle counters are based on a single-particle detection approach, wherein particles entrained in air are rapidly drawn through an intense light beam, and light scattered by single particles is sensed and used to infer particle size. Recently, this approach has been expanded to measurement of the two-dimensional angular optical scattering of single aerosol particles and the intrinsic laser-induced fluorescence (LIF) of particles, both of which may be used for additional characterization. LIF can be used in addition to (or instead of) elastic scattering. These efforts concentrate on measurement of the undispersed fluorescence of particles, and consequently only have limited potential for providing information on particle composition.
More capable techniques to measure the LIF spectra of single aerosol particles have been recently developed in order to obtain better aerosol classification. In these investigations the emphasis was on detecting biological aerosols using both cw and pulsed laser sources with wavelengths ranging from 263 nm to 488 nm. As disclosed in U.S. patent application Ser. No. 09/579,707 filed on May 25, 2000, and incorporated herein by reference, a diode-pumped, solid-state, 266 nm pulsed laser source, a Schwartzchild reflective objective with large numerical aperture for collection of fluorescence, and an intensified-CCD detector mounted on the exit port of a spectrograph, may be used to measure fluorescence spectra of single bio-aerosol particles as small as 2 micrometer diameter.
However, this significant achievement is hampered by some deficiencies. The sample cell was not airtight, so the aerosol had to be forced under positive air pressure into the sample region. Thus ambient air could not be easily sampled. Further, the requirement for high laser intensity to excite fluorescence (i.e., a tightly focused UV laser beam) and small depth-of-field of the reflective objective (to allow fluorescence light to be collected over a large solid angle and focused onto the spectrograph slit) resulted in a small sample region, which was only on the order of 20 μm in diameter. The resulting sample rate (for air containing particles to be detected) was less than 0.01 liters/min, which is impractically low for most applications.
For example, this sample rate is far too low for monitoring ambient air in buildings or in work environments.